Lactoferrin (LF) is an iron-binding glycoprotein present in secretory (exocrine) fluids such as milk. It has a variety of physiological activities such as bacteriostasis against pathogenic bacteria, adjusting function of leukocyte differentiation, build-up function of germicidal power, multiplicative function of lymphocyte and adjusting function of iron absorption. For that reason, it can be said that lactoferrin is important not only from a nutritional viewpoint but also a pharmacological viewpoint.
As a result, many attempts have previously been made to develop methods for purifying lactoferrin from milk. However, since lactoferrin has a very reactive molecular structure and interacts strongly with other milk proteins, it has been difficult to purify lactoferrin with high purity and in a high yield by a simple and easy operation.
In other words, in order to separate high-purity lactoferrin, an intricate process and a long period of time is necessary. In addition, the recovery efficiency of lactoferrin is disadvantageously low.
The scientific literature reports several of protocols for the isolation of lactoferrin from milk. A number of these involve isolation of LF from a natural source using ion-exchange chromatography followed by salt elution. Querinjean et al.(1), report isolation of human lactoferrin (hLF) from human milk on CM Sephadex C-50 followed by elution with 0.33M NaCl. Johannson employed CM Sephadex C-50 for purification of LF(2) and has also reported the use of calcium phosphate for LF purification(3). Torres et al(4), report lactoferrin isolation from guinea pig milk. The milk was pre-treated by centrifugation to remove fats and to sediment the casein. A Whatman CM-52 column was used, and lactoferrin was eluted with 0.5M NaCl/5 mM sodium phosphate, pH 7.5. Roberts and Boursnell(5), report lactoferrin isolated from defatted sow's milk. CM-Sephadex was added to an ammonium ferrous sulfate precipitate of the milk, and the bound lactoferrin was eluted with 0.5M NaCl/20 mM phosphate at pH 7 followed by a second CM-Sephadex fractionation from which the lactoferrin was eluted with 0.4M NaCl. Zagulski et al.(6), report bovine lactoferrin isolated from bovine milk. Defatted bovine milk was mixed with CM-Sephadex C-50, and lactoferrin was eluted from the column with 0.5M sodium chloride/0.02M sodium phosphate at pH 7. Moguilevsky et al.(7), report lactoferrin isolated from human milk, using CM-Sephadex chromatography and elution with 1M sodium chloride. Ekstrand and Bjorck(8), report lactoferrin isolated from human colostrum and bovine milk. Defatted bovine or human milk was acidified, adjusted to pH 7.8 and applied to a Mono S™ column. The bovine or human lactoferrin was eluted with a continuous salt gradient of 0M to 1M NaCl. The purification of human lactoferrin from bovine lactoferrin was not reported. Foley and Bates(9), report isolation of lactoferrin from human colostrum whey. The whey was mixed with a weak ion-exchange resin (cellulose phosphate) and proteins were eluted with a stepped salt and pH gradient. Lactoferrin was eluted with 0.25M NaCl/0.2M sodium phosphate at pH 7.5. Further, Yoshida and Ye-Xiuyun(10), disclosed the isolation of lactoferrin by ion exchange on carboxymethyl cation resin using 0.05M phosphate buffer at pH 7.7 with a linear gradient of 0M to 0.55M NaCl. The carboxymethyl-Toyopearl column adsorbed only lactoperoxidase and lactoferrin from the albumin fraction of bovine milk acid whey. Lactoferrin was eluted between 0.4M and 0.55M NaCl and was separated into two components; lactoferrin A and lactoferrin B. Other methods, including affinity chromatography, have also been reported. For example, in Kawakami et al.(11), affinity chromatography of LF with monoclonal antibodies to human or bovine lactoferrin was reported. Human lactoferrin was isolated from human colostrum and bovine lactoferrin from bovine milk or cheese whey. (See also U.S. Pat. No. 4,668,771, the content of which is incorporated herein by reference). Hutchens et al.(12), lactoferrin was isolated from the urine of human milk fed preterm infants with single-stranded DNA on an affinity column. Additionally, Chen and Wang(13), reported a process combining affinity chromatography with membrane filtration to isolate lactoferrin from bovine cheese whey using heparin-Sepharose to bind lactoferrin. Cheese whey was diluted with a binding buffer and added to the heparin-Sepharose material. The slurry was microfiltered, and the lactoferrin was eluted with 5 mM veronal-hydrochloride/0.6M NaCl at pH 7.4. Bezwoda et al.(14), report the use of Cibacron Blue F3GA resin for purification of LF. The purification of ferritin (Pahud et al.(15)) and heparin (Blackberg.(16)) from milk has also been reported.